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Background: Inborn errors of immunity have been implicated in causing immune dysregulation, including allergic diseases. STAT6 is a key regulator of allergic responses. Objectives: This study sought to characterize a novel gain-of-function STAT6 mutation identified in a child with severe allergic manifestations. Methods: Whole-exome and targeted gene sequencing, lymphocyte characterization, and molecular and functional analyses of mutated STAT6 were performed. Results: This study reports a child with a missense mutation in the DNA binding domain of STAT6 (c.1114G>A, p.E372K) who presented with severe atopic dermatitis, eosinophilia, and elevated IgE. Naive lymphocytes from the affected patient displayed increased TH2- and suppressed TH1- and TH17-cell responses. The mutation augmented both basal and cytokine-induced STAT6 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reversed STAT6 hyperresponsiveness to IL-4, normalized TH1 and TH17 cells, suppressed the eosinophilia, and improved the patient\"s atopic dermatitis. Conclusions: This study identified a novel inborn error of immunity due to a STAT6 gain-of-function mutation that gave rise to severe allergic dysregulation. Janus kinase inhibitor therapy could represent an effective targeted treatment for this disorder.

35 Pág. Centro de Biotecnología y Genómica de Plantas, New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell-based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1-3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo- and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2-5).

[Introduction]: CD4+ T cells with regulatory function co-expressing Foxp3 and RORγt are linked to the development of oral tolerance towards innocuous food antigens in mice. This study aimed to discern the role played by IL-6 and retinoic acid (RA) in the in vitro generation of Foxp3+RORγt+ T cells and to investigate whether such cells have suppressive properties., [Methods]: CD4+CD25- T cells isolated from the spleen of BALB/c mice, were stimulated in the presence of IL-2 alone or together with TFG-β and different concentrations of IL-6 and/or RA. Percentage of Foxp3+, RORγt+, IL-17+, Foxp3+RORγt-, Foxp3+RORγt+, and Foxp3-RORγt+ T cells within the total CD4+ T cell population, production of cytokines (IL-10 and IL-17A) and gene expression (Foxp3, Rorc, Tgfb1, Il6, Il10, and Il17) were assessed at different time points. The phenotype and ability of cells generated from CD4+CD44-CD62L+ cells in the presence of RA to suppress effector T cell proliferation was assessed., [Results]: TGF-β plus IL-6 induced the generation of Foxp3+ and double positive Foxp3+RORγt+ T cells to a higher extent than TGF-β alone at the beginning of the incubation period, although expression of Foxp3 subsequently declined. RA, added to TGF-β, increased Foxp3 and Rorc expression and Foxp3 and RORγt transcription and promoted the differentiation of Foxp3+RORγt- and Foxp3+RORγt+ cells that expressed and secreted IL-17. Foxp3+ T cells generated in vitro in presence of RA were functionally suppressive., [Conclusions]: Under the influence of IL-2 and TGF-β, suppressive Foxp3+RORγt+ T cells that express and secrete IL-17 can be produced in vitro and RA further contributes to stabilize this phenotype., This work was supported by Ministerio de Ciencia e Innovación through grants AGL2017-88964-R, FPU16/01974 (to LP-R) and JCI-2017-31345 (to SB).

The influence of gastrointestinal digestion on the immunological properties of three different nonspecific lipid-transfer proteins (nsLTPs) described in tomato fruit has been assessed using an

This article belongs to the Section Nutritional Immunology., As part of a whole egg, egg white proteins are embedded in a lipid matrix that could modify their presentation to the immune system and their allergenic properties. The present study examines the impact of the main egg lipid components, triacylglycerides and phospholipids, in the early events of sensitization to egg. To this end, BALB/c mice were exposed intragastrically to egg lipids and egg lipid fractions, alone and in mixtures with egg white proteins, and Th2-promoting and proinflammatory effects were investigated. Our results highlight that the egg lipid fraction is responsible for Th2 adjuvant effects and point at a different influence of triacylglycerides and phospholipids on the bioavailability and immunomodulating properties of egg white proteins. While triacylglycerides promote type 2 responses at the small intestine level, phospholipids reduce the solubility of EW proteins and induce Th2 skewing in lymphoid intestinal tissues, which may have a direct impact on the development of egg allergy., This study was funded by the Ministerio de Economía, Industria y Competitividad of the Government of Spain (projects AGL2017-88964-R and AGL2017-87884-R). S.B. is supported by a Juan de la Cierva Incorporación contract (Ministerio de Economía, Industria y Competitividad of the Government of Spain) and L.P.-R. is recipient of a FPU grant (Ministerio de Educación, Cultura y Deporte of the Government of Spain).

This article belongs to the Special Issue Molecular Approaches to Food Allergy Diagnosis, Treatment and Identification of Allergens., Oral food challenge (OFC) remains the gold standard for the diagnosis of food allergies. However, this test is not without risks, given that severe allergic reactions can be triggered while it is conducted. The purpose of this study is to identify potential demographic variables, clinical characteristics of the patients and biomarkers that may be associated with severe reactions during the hazelnut oral challenge test. The sample included 22 children allergic to hazelnut who underwent a tree nut skin prick test (SPT), specific IgE (sIgE) to hazelnut, component-resolved diagnosis (CRD) with different hazelnut allergens (Cor a 1, Cor a 8, Cor a 9, Cor a 11, Cor a 14), and a single-blind placebo-controlled challenge with hazelnut. A statistically significant relationship was found between the severity of the reaction and the highest values of sIgE to hazelnut, Cor a 11 and Cor a 14, cumulative symptom-triggering dose and sunflower seed sensitization. The use of the CRD is a useful tool to identify patients at higher risk of developing a severe reaction. In this pediatric population sample from Spain, storage proteins were confirmed to be most involved in hazelnut allergy and the development of severe reactions., This research was funded by SEAIC Foundation of the Spanish Society of Allergy and Clinical Immunology.

The influence of gastrointestinal digestion on the immunological properties of three different nonspecific lipid-transfer proteins (nsLTPs) described in tomato fruit has been assessed using an in vitro system mimicking the stomach and intestine digestion conditions. Tomato peel/pulp nsLTP, Sola l 3, was degraded after digestion, although the immunoglobulin E (IgE) recognition of intact protein and a 10 kDa band were still observed after 30 min of duodenal digestion in the presence of phosphatidylcholine. The tomato seed nsLTP, Sola l 7, showed a higher stability than the other seed allergen, Sola l 6, during digestion. Sola l 7 showed an IgE immunoreactive 6.5 kDa band in immunoblotting analysis, retaining up to 7% of its IgE-binding capacity in inhibition ELISA test after 60 min of duodenal digestion and keeping intact its ability to activate basophils after digestion. These results suggest that the tomato seed allergen Sola l 7 might be considered as an important allergen in the induction of allergic responses to tomato due to its high stability against gastrointestinal digestion., This work was supported by grants SAF2017-86483-R from the Ministerio de Economía y Competitividad and PI13/00928 Instituto de Salud Carlos III (ISCIII), ISCIII cofounded by Fondo Europeo de Desarrollo Regional – FEDER for the Thematic Networks and Co-operative Research Centres; RIRAAF RD16/0006/0024 and ARADyAL (RD16/0006/0001; RD16/0006/0013; RD16/0006/0014); and EAACI Exchange Research Fellowship 2016. The authors would like to thank the excellent technical support of Sara Abián. S.B. was supported by a Juan de la Cierva Incorporación contract from the Spanish Ministerio de Ciencia, Innovación y Universidades.

This study investigates the potential of a hydrolysate of ovalbumin with pepsin (OP) to preclude Th2-type immunity by the enhancement of tolerogenic dendritic cells (DCs) and regulatory T (Treg) cells. Through Toll-like receptor (TLR) stimulation, OP enhances the retinoic acid pathway on DCs by means of the induction of aldehyde dehydrogenase enzymes and transforming growth factor beta (TGF-&beta, ), and it confers upon DC the ability to upregulate interleukin 10 (IL-10) as well as other tolerance-promoting mediators downstream of TRL signalling, such as IL-27, IL-33, Notch ligands, OX40L, and the transcription factors IRF4 and IRF8. OP-conditioned DCs induce the expansion of Foxp3+ and Tr1 cells in co-culture with CD4+ T cells. Furthermore, OP directly conditions CD4+ T cells from na&iuml, ve mice, without the mediation of DCs, to express aldehyde dehydrogenase (ALDH) enzymes and, in the presence of the Th2 cytokine IL-4 and exogenous TGF-&beta, it enhances Foxp3 expression. It is noteworthy that, on CD4+ T cells isolated from egg-allergic mice, OP significantly enriches the levels of Foxp3+ and Foxp3+ ROR&gamma, t+ CD4+ T cells. In conclusion, we show that food peptides may work, analogously to microbial-driven signals, through TLRs, to promote a tolerogenic phenotype on cells of the innate and adaptive immune system, a property that is further enhanced in the context of a Th2 cytokine-rich environment.

“Torta del Casar” is a Spanish soft-ripened cheese made with sheep's raw milk and subjected to a short ripening process, which favors the growth of pathogenic microorganisms including Listeria monocytogenes. The development of strategies to control pathogens and minimize health risks associated with the presence of L. monocytogenes in these products is of great interest. In this regard, the anti-Listeria activity of a whey protein hydrolysate (ProH) alone or combined with six lactic acid bacteria strains isolated from cheese was evaluated in this study as a biocontrol strategy using a “Torta del Casar” cheese-based medium. The most active combinations of lactic acid bacteria assayed induced a reduction higher than two logarithmic units in the growth of L. monocytogenes (serotype 4b) compared to their respective control when they were co-inoculated in “Torta del Casar” cheese-based medium at 7 °C for 7 days. In addition, the observed downregulation of some key virulence genes of L. monocytogenes suggests that the strain Lactiplantibacillus plantarum B2 alone and combined with the strain Lactiplantibacillus spp. B4 are good candidates to be used as biocontrol agents against L. monocytogenes growth in traditional soft cheeses based on raw milk during their storage at refrigeration temperatures., This work has been funded by the INIA throughout the projects RTA-2017-00027-C03-03 and the Junta de Extremadura and FEDER by IB16149, GR15108 and GR18056. We thank the Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM) for supplying the hydrolysate obtained from whey proteins. I. Martín is supporting by a pre-doctoral fellowship from the Spanish Ministry of Education, Culture and Sports (FPU 16/05303).

[Purpose]: Egg yolk (EY) may play a role during the sensitizing phase of egg allergy by exerting intestinal type 2-biasing effects. We aimed to identify the mechanism and role of EY in the induction of allergy to egg white (EW)., [Results]: Administration of EW upregulated duodenal expression of factors that influence epithelial barrier integrity and function, such as Muc2 and Cldn2, type 2-promoting epithelial cytokines Il33 and Il25, DC genes Irf4 and Tnfsf4, and Th2-cytokines Il14 and Il13. EW:EY further increased the expression of Il25 and Tslp in the duodenum, Il33 and Tslp in the jejunum, and the proportion of lamina propria group 2 innate immune cells (ILC2s) over EW alone. Moreover, it distinctively enhanced the expression of Irf4 and Cd1d1 in the Peyer’s patches (PPs), and of Il6, Il33, Gata3, and Il13, both in PPs and mesenteric lymph nodes. In co-cultures of DCs and T cells, EW:EY induced a higher expression of Gata3, Il4, and Il13, secretion of IL-13 and expansion of CD4+ T cells expressing ST2, the IL-33 receptor, than EW or EY added individually., [Conclusion]: Co-administration of EY may promote sensitization to EW through activation of innate immune cells, such as IECs, DCs and ILC2s, that are central to the progress of allergies., This study was funded by the project AGL2017-88964-R and L P-R is recipient of a FPU grant (Ministerio de Ciencia, Innovación y Universidades).

[Scope] The mechanism through which peptide-based immunotherapy provides effective desensitization toward food allergy is investigated., [Methods and results] Ex vivo experiments are conducted with intestinal epithelial cells (IECs), dendritic cells (DCs), and T cells from mice sensitized to egg white (EW) and either left untreated or tolerized by the oral administration of a hydrolysate of ovalbumin with pepsin (OP). IECs from EW-sensitized mice upregulate Il33 and Tslp to a higher extent than those from tolerized mice and induce bone marrow (BM)-DCs to express Tnfsf4 and produce pro-inflammatory cytokines. On the other hand, incubation with OP upregulates Aldh1a1 in IEC cultures and BM-DCs conditioned with supernatants of OP-pulsed IECs also overexpress Aldh1a2 and Tgfb1. DCs from tolerized mice, in co-culture with CD4+ T cells from sensitized mice, reduce the secretion of IL-5, IFN-γ , and IL-17, following stimulation with EW, to a level similar than DCs from sham-sensitized mice. Furthermore, incubation with OP of DCs and CD4+ T cells, regardless of the mouse sentitization status, promotes the secretion of TGF-β and the generation of Foxp3+ RORγ t + cells, [Conclusion] OP induces the expression of aldehyde dehydrogenase enzymes in cells of the innate immune system and the development of Foxp3+ RORγ t + T cells., This study was funded by the project AGL2017-88964-R and L.P.-R. is recipient of an FPU grant (Ministerio de Ciencia, Innovacion y Universidades).

Resumen del trabajo presentado al American Academy of Allergy, Asthma and Immunology (AAAAI) Annual Meeting, celebrado en San Francisco, California (USA) del 22 al 25 de febrero de 2019., [Rationale] Oral immunotherapy (OIT) with peptides has proved more effective than that with intact allergens in providing desensitization against food allergy. The mechanism by which food peptides confer higher protection was investigated., [Methods] BALB/c mice sensitized to egg white (EW) with the aid of cholera toxin (CT) were subjected to OIT with intact and pepsin-hydrolysed EW (EP). Changes in the caecal microbiota and its metabolism, generation of barrier-protective responses in the small and large intestine and expansion of regulatory T cells were studied., [Results] Treatment with EP was superior to that with EW in terms of reduction of anaphylaxis and levels of specific antibodies. OIT with EP, but not with EW, modulated the microbiota by restoring the levels of some members of the order Clostridiales (clusters IV and XIVa) that were affected by CT-induced sensitization. Mice treated with EP exhibited upregulated intestinal expression of Il22 and Muc2, which encode for factors that contribute to reinforce the epithelial barrier function, as well as Aldh1a1, Aldh1a2 and Tfgb1, that take part in the conversion of vitamin A into retinoic acid. Tolerance induced by EP paralleled the enhanced expression of Foxp3, Rorc and Il17 in non-lymphoid and lymphoid tissues and the development of Foxp3+ cells that simultaneously expressed RORγt., [Conclusions] The benefits of peptide OITwere associated to vitamin A metabolism and development of immune cells that depend on RORγt for their transcriptional regulation.

This study investigates the mechanism by which food peptides are more effective than intact allergens in providing desensitization against food allergy. BALB/c mice sensitized to egg white (EW) were subjected to oral immunotherapy (OIT) with intact and pepsin-hydrolysed EW (EP). Treatment with EP was superior to that with EW in terms of reduction of anaphylaxis and levels of specific antibodies. OIT with EP, but not with EW, modulated the microbiota by restoring the levels of some members of the order Clostridiales (clusters IV and XIVa) that were affected by sensitization. Mice treated with EP exhibited upregulated intestinal expression of Il22 and Muc2, which encode for factors that contribute to reinforce the epithelial barrier function, as well as Aldh1a1, Aldh1a2, Csf2 and Tfgb1, that take part in the conversion of vitamin A into retinoic acid. Tolerance induced by EP paralleled the development of Foxp3 cells that simultaneously expressed RORγt., This study was funded by the projects AGL2017-88964-R and AGL2016-75951-R, and L P-R is recipient of a FPU grant (Ministerio de Ciencia, Innovación y Universidades).

[Scope]: Egg is the second most frequent source of allergic reactions in children. Egg yolk (EY) amounts to one-third in weight of a fresh whole egg, but its contribution to egg allergy has not been investigated in depth. This study assesses whether EY influences the capacity of egg white (EW) to sensitize and trigger allergic responses. [Methods and results]: BALB/c mice were exposed to EW, EY, and their mixture, using models of orally (with and without adjuvant) and adjuvant-free intraperitoneally induced allergy. In vitro assays were also conducted to examine epithelial and dendritic cell (DC) functions. Results showed that EY played a role during the sensitizing phase of allergy. EY exerted local Th2-biasing effects through the upregulation of intestinal IL-33 expression and it also favored Th2 polarization directly during DC presentation of allergens to T cells. [Conclusion]: The results obtained reveal that EY provides Th2-adjuvant stimuli to the immune system that may increase the susceptibility to develop egg allergy. The joint administration of EW and EY may be a trigger for initiation or maintenance of egg allergy with implications in prevention strategies regarding egg introduction in the diet of susceptible children., This study was funded by the project AGL2014-59771-R (MINECO, Spain).

This study assesses to what extent technological processes that lead to different degrees of denaturation of egg white proteins affect their allergenicity. We focused on heat (80 °C, 10 min) and high-pressure (400 MPa and 37 °C, 10 min) treatments and used a BALB/c mouse model of food allergy. Oral sensitization to egg white using cholera toxin as adjuvant induced the production of IgE and IgG1 isotypes and led to severe clinical signs following challenge with the allergen. Extensive protein denaturation caused by heat treatment increased its ability to induce Th1 responses and reduced both its sensitizing and eliciting capacity. Heated egg white stimulated the production of IgE over IgG1 antibodies directed, at least in part, toward new epitopes exposed as a result of heat treatment. Conversely, partial denaturation caused by high-pressure treatment increased the ability of egg white to stimulate Th2 responses and its allergenic potential., This work was supported by MINECO (Project AGL2014-59771R and contract of A.P.-T.) and MECD (through D.L.-O. contract).